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Abstrak :
[ABSTRAK
Pendahuluan: Infeksi dengue merupakan salah satu penyakit endemik di daerah tropis dan subtropis yang disebabkan oleh virus dengue (DENV). Hingga saat ini belum ada antiviral yang efektif untuk infeksi dengue. Penyebaran dan sirkulasi serotipe DENV berfariasi di setiap lokasi geografi, hal ini menyulitkan dalam melakukan evaluasi vaksin DENV. Oleh karena itu perlu dikembangkan kandidat vaksin DENV menggunakan strain Indonesia supaya dapat memberikan proteksi maksimal. Pada peneltian ini dikembangkan kandidat vaksin DNA tetravalen DENV berbasis gen prM-E DENV strain Indonesia. Metode: Konstruksi plasmid rekombinan kandidat vaksi dilakukan dengan cara menyisipkan gen prM-E setiap serotipe DENV ke dalam vektor pUMVC4a. Gen prM-E DENV merupakan strain Indonesia, yang diamplifikasi dari serum pasien yang terinfeksi dengan virus ini. Kemampuan plasmid rekombinan mengekspresikan protein prM-E DENV diuji di sel mamalia. Kemampuan kandidat vaksin menginduksi respon imun humoral dievaluasi secara monovalen dan tetravalen di mencit jenis ddY. Titer IgG anti dengue diperiksa menggunakan teknik ELISA, sedangkan titer antibodi netralisasi di tentukan dengan uji FRNT. Proteksi vaksin terhadap mencit yang diimunisasi dievaluasi dengan melakukan uji tantang menggunakan sel K562 yang diinfeksi DENV-2. Viremi virus di tentukan dengan menggunakan teknik foccus assay. Hasil: Konstruksi plasmid rekombinan kandidat vaksin DENV-1 dan DENV-3 sudah berhasil dilakukan. Plasmid dapat mengekspresikan protein prM-E DENV di sel mamalia, namun karakteristik dan kinetik protein masih belum dapat diketahui dengan jelas. Keempat kandidat vaksin DNA yang sedang dikembangkan dapat menginduksi respon imun, baik secara monovalen maupun tetravalen. Imunisasi secara tetravalen dapat memberikan proteksi pada mencit yang diuji tantang dengan sel K562 yang diinfeksi dengan DENV-2.;

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ABSTRACT
Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate. Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay. Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2.;Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate. Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay. Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2., Introduction: Dengue infections are caused by dengue viruses (DENV) and are endemic in tropical and subtropical regions. At present, there is no effective antiviral treatment for dengue infection. Distribution and circulation of DENV serotypes varies by geographic location, it is difficult to evaluate DENV vaccine. Therefore, it is necessary to develop a vaccine candidate DENV using Indonesian strains in order to provide maximum protection. However, in this study, we constructed a recombinant plasmid-based prM-E gene from the Indonesia strain as a DENV DNA vaccine candidate. Methode: The recombinant plasmid was prepared by inserting the prM-E gene from each DENV serotypes into the plasmid backbone pUMVC4a. prM-E gene an Indonesia strain, which was amplified from patient sera infected with DENV. The ability of the recombinant plasmid expressing the prM-E DENV protein tested in mammalian cells. The ability of candidate vaccines induce humoral immune responses were evaluated monovalent and tetravalent in ddY mice. IgG titers of anti-dengue examined using ELISA technique, while neutralizing antibody titers determined with FRNT test. Vaccine protection against the immunized mice was evaluated by conducting challenge test using K562 cells infected by DENV-2. Viremia was determined by using the foccus assay. Result: Construction of recombinant plasmid vaccine candidate DENV-1 and DENV-3 was successfully performed. Plasmids can express prM-E DENV proteins in mammalian cells, but the characteristics and kinetics of protein still can not clearly known. Fourth DNA vaccine candidate that is being developed to induce an immune response, either monovalent or tetravalent. Tetravalent immunization may provide protection in mice challenged tested with K562 cells infected with DENV-2.]

Abstrak :
Penyakit dengue merupakan penyakit yang disebabkan oleh virus dengue dengan empat serotipenya yaitu DENV-1, 2, 3, dan 4. Untuk mengurangi terjadinya penyakit dengue dari keempat serotipe yang ada, maka perlu dirancang vaksin peptida yang bersifat tetravalen dan mencegah infeksi semua serotipe dengue menggunakan protein E (envelope) DENV-3 sebagai backbone. Prediksi epitope sel T protein E DENV1-4 menggunakan server MULTIPRED dengan HLA kelas II DR dan algoritma Hidden Markov Model (HMM), dan prediksi epitope sel B protein E DENV-3 menggunakan Conformational Epitope Prediction (CEP) server. Perancangan vaksin dilakukan dengan mensubstitusikan epitope sel T highest binders protein E DENV-1,2, dan 4 ke dalam epitope sel T non binders backbone protein E DENV-3. Dengan memvariasi posisi epitope pensubstitusi maka diperoleh enam rancangan vaksin peptida dengue yang bersifat tetravalen. Hasil BLASTp menunjukkan keenam rancangan vaksin mempunyai 93% identity dengan protein E DENV-3 native. Struktur tersier vaksin diprediksi melalui homology modeling. Dari hasil analisis struktur tersier diperoleh empat rancangan vaksin yang terbaik, yaitu HMM1-2-4, HMM1-4-2, HMM2-1-4, dan HMM4-1-2 dengan persentase residu non glisin pada disallowed region berkisar 2% dan score VAST 61,5. Prediksi sistem ekspresi protein menghasilkan sel serangga sebagai host yang sesuai untuk mengekspresikan keempat vaksin tersebut. Rancangan DNA rekombinan menggunakan plasmid pBlueBac4.5 sebagai vektor transfer baculovirus dan insersi sekuens DNA vaksin menggunakan enzim restriksi BamH1 dan EcoR1. Prediksi modifikasi post translation menunjukkan hanya modifikasi N-glikosilasi yang paling mungkin terjadi terhadap protein vaksin dalam sel serangga.

Abstrak :
Epidemi dengue menyerang berbagai negara di dunia, khususnya negara-negara tropis dan subtropis. Infeksi dengue disebabkan oleh dengue virus (DV) yang memiliki serotype (DV1, DV2, DV3, dan DV4). Penelitian ini bertujuan untuk mendapatkan desain sekuens vaksin dengue yang bersifat tetravalen melalui studi bioinformatika. Protein envelope (E) pada keempat serotype DV digunakan untuk merancang sekuens vaksin. Multiple alignment digunakan untuk melihat similaritas 102 intra-serotype DV. Perwakilan dari tiap serotype DV diambil berdasarkan hasil multiple alignment, tingkat insedensi, dan letak geografis penemuan DV. Epitope ditentukan melalui server MULTIPRED dengan dua metode algoritma. Tiga epitope dari masing-masing metode algoritma, disubsitusi ke dalam backbone DV2 sehingga didapatkan dua rancangan sekuens vaksin dengue (vaksin A dan vaksin H). Rancangan sekuens tersebut dicari kesamaan strukturnya melalui server BLAST. Hasil analisis BLAST menghasilkan 91% identitas, 895 bits score, 0.0 E-value untuk vaksin A dan 92% identitas, 890 bit score, 0.0 E-value untuk vaksin H. Berdasarkan hasil analisis BLAST, kedua rancanan vaksin dengue tersebut memiliki struktur dan folding akhir yang serupa dengan backbone DV2.